Combination of tumor-associated surface protein antigens and tumor-associated sugars in the treatment and diagnosis of cancer

ABSTRACT

The invention relates to a kit for the combined use for the treatment of cancer patients, which set comprises an antigen of a cellular surface protein, or an antibody directed against the cellular surface protein, and an antigen of an aberrant glycosylation, or an antibody directed against the aberrant glycosylation. This kit is destined both for the immunotherapeutic and the diagnostic application. The invention further relates to a selection method for selecting suitable tumor-specific antigens with the assistance of this kit and corresponding specific antibody preparations.

The invention relates to a set for combined application for thetreatment of cancer patients, as well as its diagnostic use. Also, amethod of screening of neoepitopes and their use is disclosed.

Cancer is a wide-spread disease and is lethal in many cases. The therapyof cancer usually comprises the removal of a solid tumor, and a furthertreatment which is to prevent and reduce, respectively, metastases.Besides surgery, the standard therapies include chemotherapy andradiation therapy. Despite the comprehensive therapy which ofteninvolves severe side effects, the success of treatment is insufficient.Most frequently, cancer forms with epithelial tumors occur, which interalia concern breast, stomach intestines, pancreas, lungs, prostate andovaries. The relapse rate in intestinal cancer is approximately 45%.Mestastatic epithelial cancer is considered to be nearly incurable.Therefore, in the treatment of cancer patients it is important toprevent, and reduce, respectively the formation of metastases.

Tumor cells are capable of disseminating from primary tumors in bodyliquids and other organs. These disseminated tumor cells may be in theirdormant state and often cannot be attacked by a chemotherapy(radiotherapy). Such a treated patient seems to be in a cured state,which is described as “minimal residual disease”. Dormant tumor cells,however, have a potential of forming metastases if they become growingand metastasizing cells.

Immunotherapy constitutes an innovative possible treatment of cancerpatients. Both active and also passive immunotherapy are acknowledgedmeasures for supporting the immune system.

The adaptive immune system of humans consists of two essentialcomponents, the humoral and the cellular immunity. The adaptive immuneresponse partially is based on the clonal selection of B- andT-lymphocytes and in principle allows for the recognition of any desiredantigen as well as for the build-up of an immunological memory. Thesecharacteristics of the adaptive immune system are generally usefullyaddressed in vaccinations.

Each B-cell produces an antibody with a defined binding specificity.This antibody is also present as a specific receptor in the membrane ofthe B-cell producing it. The humoral immune response against antigensrecognized as foreign is based on the selective activation of thoseB-cells which produce such antibodies that can bind to an epitope of therespective antigen. For the antibody diversity, DNA rearrangements inthe course of B-cell differentiation play a decisive role.

There are several possible ways of interfering in the immune system. Oneapproach of relatively specifically destroying tumor cells is thepassive immunotherapy with antibodies directed against tumor-associatedantigens (TAA) (Immunology Today (2000), 21:403-410; Curr. Opin.Immunol. (1997), 9:717). Another approach of destroying tumor cells isthe active vaccination which triggers an immune response against TAA.This immune response thus is also directed against the correspondingtumor cells (Ann. Med. (1999), 31:66; Immunobiol. (1999), 201:1).

1. Passive Antibody Therapy:

For therapeutic purposes, it is possible to supply to an organismantibodies required for a certain function within this organism. Thistype of application is called passive immunotherapy, and it can be usedin various medical indications, e.g. in the immunotherapy of cancer(Immunol. Today (2000), 21:403), intoxications (Toxicon (1998), 36:823;Therapie (1994), 49:41) and infections (Clin. Infect. Dis. (1995),21:150). In these cases, antibodies can be used which either have beenderived from appropriately immunized animals or can be recovered fromcells by various biological or molecular-biological techniques (e.g.hybridoma technique, phage-display technique, etc.) via theimmortalization of immunoglobulin genes.

2. Active Immunization:

To modulate the immune system, an immunization with antigens can beused. Antigens are molecules, molecule complexes or whole organisms towhich antibodies can bind. Not all the antigens induce an immuneresponse, i.e. not all the antigens are immunogenic. Certain smallmolecules are not registered by the immune system (haptens), suchsmaller molecules can be presented to the immune system in suitableform, and thus be made immunogenic. Such a method is the coupling of thehapten to an immunogenic molecule, a so-called carrier molecule. For anactive immunization, also antibody preparations can be used, asdescribed in EP 1140168.

Tumor cells can be attacked by the immune system only to a limitedextent, since they are hardly different from normal cells and specificantibodies therefore are missing. Much research is directed to theidentification of suitable targets, i.e. target antigens, for thepreparation of tumor-specific antibodies. The immunotherapy for thetreatment of cancer then either comprises the passive therapy by thedirect administration of the specific antibodies, or the activevaccination with suitable antigen-targets for stimulating the immunesystem and generating the specific antibodies in vivo.

Certain TAAs are defined as relevant “targets” for the development ofimmunotherapeutic agents for the prophylaxis and/or treatment of cancer.TAAs are structures which preferably are expressed on the cell membraneof tumor cells, thereby allow for a differentiation relative tonon-malignant tissue, and thus can be viewed as targets for thediagnostic and therapeutic applications of specific antibodies.

Beside other physiological characteristics which distinguish them fromnormal cells, cancer cells practically always have a changed type ofglycosylation (Glycoconj. J. (1997), 14:569; Adv. Cancer Res. (1989),52:257; Cancer Res. (1996), 56:5309). Even though the changes aredifferent from tissue to tissue, it can be found that an aberrantglycosylation is typical of cancer cells. In most instances, the changedglycosylation is presented at the surface of cells in the form ofglycoproteins and glycolipids. These changed sugar structures thereforecan be designated as TAAs which in many cases are sufficientlytumor-specific, i.e. they rarely appear in “normal cells”. In manyinstances, the cells do not have a uniform glycosylation, and neither dothe tumor cells, i.e. there exist various glyco-forms of complex glycanchains on a cell (Annu. Rev. Biochem. (1988), 57:785).

In the course of the discovery and the subsequent characterization ofvarious TAAs it has been found that they have important functions forcancer cells. They allow the degenerate cells to have propertiescharacteristic of the malignant phenotype, such as, e.g., an increasedadhesion capacity, or an increased uptake of growth factors, which arehighly important for establishing metastases. However, in certainstages, such antigens may very well also be expressed on normal cellswhere they are responsible for normal functions of these cells. Anexample of this is the Lewis Y carbohydrate antigen which appears on theplurality of tumors of epithelial origin, but also plays an importantrole during the fetal development of epithelial tissues. It has beenshown that the expression of this antigen in lung cancer is associatedwith an unfavorable prognosis, since Lewis Y positive cancer cellsapparently have a higher metastatic potential (N. Engl. J. Med. 327(1992), 14).

In EP 0 528 767, the use of a humanized anti-Lewis Y antibody for thetreatment of epithelial cancer has been described.

Among the further known tumor-associated carbohydrate structures, thereare, e.g., all those Lewis antigens which are increasingly expressed inmany types of epithelial cancers. Among them are Lewis x- and Lewisb-structures in addition to Lewis y-structures, as well as sialylatedLewis x-structures. Other carbohydrate antigens are GloboH-structures,KH1, Tn antigen, sialylTn, TF antigen, the alpha-1,3-galactosyl epitope(Elektrophoresis (1999), 20:362; Curr. Pharmaceutical Design (2000),6:485, Neoplasma (1996), 43:285).

Other TAAs are proteins which are particularly highly expressed bycancer cells, such as, e.g. CEA, TAG-72, MUC1, Folate Binding ProteinA-33, CA125, EpCAM, HER-2/neu, PSA, MART, etc. (Sem. Cancer Biol.(1995), 6:321). Relevant TAAs often are surface antigens of epithelialcells which occur in larger numbers in growing cells, such as fetaltissue, and also in tumor tissue. A special group of TAAs are involvedin the adhesion processes of the epithelial cells. Among the cellularadhesion proteins which are over-expressed on tumor cells are EpCAM,NCAM and CEA.

In Austrian application A 744/2002, an immunogenic antibody having atleast two different epitopes of a TAA has been described. A preferredantibody comprises at least one epitope of EpCAM and one epitope ofLewis Y.

According to Allergol. et Immunopathol 25, 4 (176-81), 1997, the MUC-1gene product, the polymorphous epithelial mucine, and the development ofspecific monoclonal antibodies is described. As the target for the TAAmucine, both the peptide sequence and also specific carbohydrates areexamined.

Direct therapeutical applications of antibodies against TAA are based onpassive immunotherapies, i.e., a specific antibody is systemicallyadministered in a suitable amount to cancer patients, and has animmunotherapeutic effect. The biological half-life of such agents willdepend on their structure and is limited. Therefore, it is necessary tocarry out repeated applications. When using xenogenic antibodies (e.g.monoclonal antibodies, MABs, for example of murine origin) however, thiscan lead to undesired immune reactions which may neutralise a possibletherapeutic effect and may cause dangerous side effects (eg. immunecomplex formation with kidney failure, anaphylactic reactions).Therefore, such immunotherapeutic agents can be administered for alimited time only.

A better tolerance is obtained by reducing the xenogenic structures ofthe antibody and by introducing human structures, e.g. with chimeric orhumanized antibodies. Also systems for producing specific humanantibodies are developed. Thus, according to the prior art, certain celllines, organisms or transgenic animals can produce human monoclonalantibodies.

The invention has as its object to improve the immunotherapeutictreatment of cancer patients by the selection of suitable targetantigens and the accompanying diagnostics

According to the invention, this object is achieved by the subjectmatter of the claims.

According to the invention, a kit is provided which is suitable for thecombined application for the treatment of cancer patients. The kitcomprises the components

-   -   a) an antigen of a cellular surface protein, or an antibody        directed against the cellular surface protein, and    -   b) an antigen of an aberrant glycosylation, or an antibody        directed against the aberrant glycosylation.

Besides other physiological characteristics which distinguish them fromnormal cells of the respective tissue, cancer cells practically alwayshave a changed type of glycosylation as has been mentioned before(Glycoconj. J. (1997), 14:569; Adv. Cancer Res. (1989), 52:257; CancerRes. (1996), 56:5309). Even though the changes are different from tissueto tissue, it can be found that an aberrant glycosylation is typical ofcancer cells (as compared to the corresponding normal cells in thistissue). In most instances, the changed glycosylation will be presentedat the surface of the cells in the form of glycoproteins andglycolipids. These changed sugar structures therefore can be designatedas TAAS, which in many instances are sufficiently tumor-specific, i.e.they rarely occur in “normal” cells. In many instances, the cells, andalso the tumor cells, do not produce a uniform glycosylation, i.e. thereexist various glycoforms of complex glycan chains on one cell (Annu.Rev. Biochem. (1988), 57:785).

The combination of the active substances thus either relates to anantigen-antigen, an antigen-antibody or an antibody-antibodycombination. With the inventive kit, effective immunotherapies directedagainst tumor-specific target antigens are rendered possible.

Surprisingly, it has been shown that the common, simultaneous, parallelor consecutive specific formation of the immune complexes of antibodieswith the antigens of the components of the kit contributes particularlyto the positive course of the disease in a cancer patient. Inparticular, it has been found that cancer patients who have both targetantigens together on solid tumors have an unproportionately worseprognoses of the survival time as compared to patients with only one ofthe target antigens. The immunotherapeutic treatment with the inventivecombination of the tumor-associated components therefore is consideredas particularly effective for increasing the survival time or therelapse-free time, respectively.

The target antigens of the component a) preferably constitute at leastone epitope of a cellular adhesion protein, and preferably they areselected from the homophilic adhesion proteins of epithelial tumorcells. They have the properties that they can bind to the same surfaceproteins of other tumor cells and thus are capable of forming a cellagglomerate. Among them are antigens which are particularly derived fromthe EpCAM antigen, the EpCAM molecule itself or epitopes, parts ormimics thereof. A particularly good immunogen for the activeimmunization with an antigen of component a) is an immunogenic antibodyas described in EP 1140168 B1. Further particularly preferred antigensof cellular adhesion proteins have been derived either from NCAM or fromCEA.

According to a further preferred embodiment, the antigen of component a)has at least one epitope of a surface receptor, in particular a receptormolecule selected from the group of the EGF receptor family, among themthe EGF receptor and Her-2/neu receptor, CD55 receptor, transferrinreceptor and P-glycoprotein. Yet, moreover, also antigens with epitopesof a mucine, in particular MUC1, or CA125, can be used according to theinvention.

Among the epitopes relevant for component a), preferably there is atleast one epitope of a human antigen selected from the group of peptidesor proteins with regulating function for cellular adhesion processes andreceptor functions. Besides the already mentioned antigens, theyparticularly include also T-cell peptides which preferably have beenderived from the TAAs.

An antigen of component b) of the inventive kit is directed to theimmunotherapy against an epitope of a carbohydrate. Carbohydrateepitopes preferred according to the invention are tumor-associated,aberrant carbohydrate structures, such as the Lewis antigens, e.g. Lewisx-, Lewis b- and Lewis y-structures, as well as sialylated Lewisx-structures. Moreover, also GloboH structures, KH1, Tn antigen, TFantigen, the alpha-1,3-galactosyl-epitope are preferred carbohydrateantigen structures within the scope of the present invention.

A particularly good target for component b) is the Lewis Y antigen.Particularly preferred is the use of a humanized antibody, such asdescribed in EP 0 528 767. This antibody recognizes Lewis Y antigen ontumor cells or on surface receptors of tumor cells, respectively, and issuitable for the passive immunotherapy.

It has been found that the surface receptors of a tumor cell areequipped with an aberrant glycosylation and form relevant epitopes asdefined by the invention. This primarily relates to the EGF receptorfamily, among them the EGF receptor or Her-2/neu receptor. Additionally,also neoepitopes being formed by the aberrant glycoslylation of anantigen of a cellular surface protein with a carbohydrate structure areincluded according to the invention. The inventive combination of thetarget antigens which are derived from these glycoproteins, optionallywith further antigens of the aberrant glycosylation, therefore isparticularly preferred.

By the immunotherapy with the target of the aberrant glycosylation,practically all tumor-specific receptors which are characterized by thisaberrant glycosylation are blocked. Among them are, e.g., all thereceptors of the EGF-receptor family, the CD55 (791Tgp72/DAF—decayaccelerating factor) receptor, the transferrin receptor, and theP-glycoprotein.

It has also been found that antibodies which are directed against anaberrant glycosylation bind in a functional manner to several receptorsof the family of the EGF receptors and thus the signal cascade forinducing the cell growth can effectively be blocked. In Austrianapplication A 995/20 02 it could be demonstrated that it was possible tofunctionally bind in particular the erk1 and erk2 isoforms of the MAPkinase by means of the inventively used antibodies. The binding of thegrowth factors to the receptors was thereby prevented or reduced,respectively. This treatment is more specific as compared toimmunotherapy using antibodies against the proteinaceous extracellularpart of the EGF receptor, since the unusual tumor-associatedcarbohydrate structures are missing on the EGF receptors of normalcells. On the other hand, the treatment is more universal, sincesimultaneously different receptors having the same aberrantglycosylation are blocked.

By the inventive use of the immunotherapy, directed against an aberrantglycosylation, thus also the mitogenic stimulation of a cancer cell byEGF or heregulin is prevented. The specific binding of the antibodies toa tumor-associated glycosylation of cancer cells blocks the interactionof the receptors of growth factors with their physiologic ligands andinhibits the signal transduction through these receptors and thus, thecell growth.

At the same time, such an antibody can specifically attack the tumorcell by its effect within the humoral and cellular immune system. Tumorcells which express the EGF receptor or receptors of the EGF receptorfamily, respectively, according to the invention are specifically boundand can be lysed or be blocked in growth.

The particularly preferred inventive combination of the target antigensrelates to an epitope of the EpCAM molecule or of the Her-2/neu receptorfor the component a), and an epitope of the Lewis Y molecule for thecomponent b).

The target antigens are either directly affected by the specificimmunotherapy, or indirectly, by the binding of TAA which substantiallynegatively affect a function of the target antigens. Target antigensparticularly comprise epitopes of proteinaceous antigens, glycoproteinsor carbohydrate antigens. As a rule, it can be assumed that by aproteinaceous antigen a polypeptide of at least five amino acids is tobe understood.

Preferred epitopes are derived from antigens which are specific ofepithelial tumors and increasingly occur e.g., in breast cancer, cancerof the stomach and intestines, such as colon and rectal, the prostate,pancreas, ovaries and the lungs. Among the preferred epitopes are thosewhich primarily induce a humoral immune response, i.e. a specificantibody formation, in vivo. On the other hand, also those antigens canbe chosen as epitopes as defined by the invention which generate aT-cell specific immune response. Among them also intracellularstructures or T-cell peptides can be found. Suitable epitopes areexpressed at least in 20%, preferably at least in 30% of the cases bytumor cells of a certain type of cancer, more preferred in at least 40%,in particular in at least 50% of the patients.

Methods of finding suitable antigen structures, modelling and producingthe TAA-derived peptides, polypeptides or proteins, or the nucleic acidscoding therefor, furthermore, lipoproteins, glycolipids, carbohydratesor lipids are known to the person skilled in the art and can be providedfor the respective tumor-specific structure without undue experimentalexpenditures. Methods for conjugating or derivatizing such structureswhich likewise are suitable according to the invention are also known.Furthermore, the methods of producing the specific antibodies that aresuitable according to the invention are known.

For selecting suitable antigenic structures, or corresponding antibodiesor immune complexes, respectively, according to the invention a suitableantigen and/or a corresponding antibody is selected by an immunologicalscreening method. The method according to the invention for theimmunological selection of a tumor-specific target antigen or ofantibodies directed against the target antigen employs a diagnosticagent. According to the invention, this agent comprises the activesubstances of the afore-mentioned components a) and b) which both formimmune complexes with an immunotherapeutic candidate eithersimultaneously or independently of each other.

Screening may be carried out with the assistance of known methods, amongthem phage display methods and hybridoma technology, and theimmunoreagents corresponding to components a) and b), or also withqualitative or preparative methods for the selective binding of thecandidate antigens to the immune reagents. An appropriate screening forthe first time also allows for the selection and the preparation of aneoepitope which is just formed by the glycosylation of an antigen ofcomponent a) with a carbohydrate structure of component b). It has beenfound that this neoepitope is then expressed by tumor cells if a cancerpatient has an unproportionately unfavorable prognosis. According to theinvention, it has now become possible for the first time to find thetumor-specific and relevant neoepitopes with the assistance of ascreening method, and to develop appropriate immunotherapies. After theneoepitope has been characterized, suitable antibody preparations can beprepared which will recognize just this neoepitope, and preferably willnot recognize one or both of the individual antigens of components a)and b). The immunotherapy with the target of the neoepitope has beenimproved insofar as epithelial cells of normal tissue will not beaffected, but merely the tumor cells.

Examples of such neoepitopes are epitopes which are formed by theglycosilation of an EpCAM protein or a Her-2/neu receptor with Lewis Ycarbohydrate or appropriately sialylated glycoproteins. When antibodieswith a specificity for these neoepitopes are produced and prepared, theypreferably do not bind to the de-glycosylated proteins nor do they bindto the carbohydrate motif on structurally different proteins. It isprecisely these antibodies which preferably are suggested as monoclonalantibodies for the passive immunotherapy so as to avoid unspecificinteractions and side effects. The identification of the neoepitopes canalso be the basis for the development of vaccination antigens, bypresenting an immunogen with exactly this epitope. This epitope or amimic of the epitope can be produced easily from appropriate peptidelibraries or by anti.idiotypic antibody techniques or also as aderivative, e.g. a fragment, of a naturally occurring antigen. On thebasis of the selected neoepitope, a preparation of an antigen isobtainable which has exactly this neoepitope or the mimic thereof, eg ananti-idiotypic antibody, mimotope. Such antigen preparations arevaluable active substances for the active immunization of cancerpatients or they can also be employed as a diagnostic preparation.

According to the invention, e.g. a tumor cell line which expresses aLewis Y glycosilated Her-2/neu receptor is used for producing therespective antibodies. With a selection method that uses the individualimmunoreagents Lewis Y, Her-2/neu and a Lewis Y glycosylated .Alternatively, Lewis Y can be synthetically produced. Her-2/neu thoseantibodies are recovered which merely bind to the combination antigen,yet not to one of the separate antigens. These antibodies can be thebasis for the development of the neoepitope-specific mimics.

According to the invention, hybridomas can be recovered from immunizedmice, the selection of the positive clones being effected by adifferential screening with the components of the inventive kit. It willthen be possible to identify a neoepitope by a usual epitope mappingmethod in that the specific antibodies bind to the glycosylated surfaceprotein, yet not to the individual components a) or b). From a positiveclone, an anti-neoepitope monoclonal antibody can then be obtained. Apreparation of such an antibody or a derivative thereof is, e.g.,suitable for the passive immunotherapy or for diagnostic use.

In a particular embodiment, for at least one of the components a) and b)an antibody mixture is provided of different antibodies with aspecificity for one or more of the antigens from a) or b). Inparticular, it is possible to provide antibody mixtures as arepresentative panel having specificity for at least two equal ordifferent epitopes of an adhesion protein, such as EpCAM, or anaberrant-glycosylation, e.g. of the Lewis carbohydrate antigens.

According to the invention, the immunotherapy is carried out with apharmaceutical preparation which comprises either both components, i.e.antigens and/or antibodies of the kit together or as separatepreparations, in pharmaceutically acceptable form. A kit according tothe invention thus preferably comprises the components a) and b) in onepreparation each or in one single pharmaceutical preparation which issuitable for immunotherapy. Alternatively, when using the neopetipoeaccording to the invention, only one component might be useable.

The kit according to the invention can be used both for an activeimmunotherapy and also for a passive immunotherapy, or for thecombination of the active and passive immunotherapy, respectively.Accordingly, the inventive pharmaceutical preparations, or theircomponents, respectively, are preferably formulated as vaccines and/oras an intravenously tolerable preparation.

A medicament used according to the invention for the passiveimmunotherapy preferably is provided in a suitable formulation.Preferred are such formulations with a pharmaceutically acceptablecarrier. The latter comprises, e.g., auxiliary substances, buffers,salts and preserving agents. Preferably, a ready-to-use infusionsolution is provided.

Since an antibody.is comparatively stable, medicaments based onantibodies or their derivatives have the substantial advantage that theycan be put on the market as storage-stable solutions or as a formulationin a ready-to-use form. The latter is preferably storage-stable atrefrigerating temperature up to room temperature.

The medicament formulated for intravenous administration may, however,also be stored in frozen or lyophilized form and may be thawed orreconstituted, respectively, upon demand.

For the passive immunotherapy, and thus for binding the relevant surfaceantigens to tumor cells, usually a high dose of at least 50 mg,preferably at least 100 mg, most preferred at least 200 mg, isadministered per patient. The maximum dose is limited by thetolerability of the antibody and will depend on its specificity andavidity. Humanized antibodies and human antibodies, respectively, arethe best tolerable. A dose of up to 1 g or in some cases of up to 2 gper patient and treatment may very well be advantageous.

The usual treatment for the passive immunotherapy comprises repeatedinfusions at regular intervals, e.g. weekly, for a period of time offrom 6 to 24 weeks, at a dose ranging from 1 to 10 mg/kg, preferablyranging from 2 to 6 mg/kg. An antibody preparation can also beadministered locally, i.e. to the tumor tissue and/or intraoperativelyinto the wound region after removal of a tumor. Various modes ofadministration may appropriately be combined, e.g. an i.v. infusionshortly before surgery, as well as a local dose directly into the woundregion during surgery. Practical application means for the localizedadministration comprise, e.g., ready-to-use applicating means, such ascatheters, syringes or sprays, suitable for distributing liquidmedicaments.

The i.v. treatment is preferably repeated at certain time intervals,corresponding to the half-life of the antibody used which usually is inthe range of from 5 to 30 days. By a special derivatization, eg.pegylation of the antibody it is possible to lengthen the half-life toup to several months, and to thereby lengthen the treatment intervalsaccordingly.

The concentration of the active substance of the medicament will dependon its tolerability. A particularly well tolerated preparation based ona humanized antibody can be administered directly to the patient at ahigh concentration and without being further diluted. By the preferredconcentration in the range of from 0.1% to 10%, preferably 1% to 5%, itis possible to keep low the administered volume and the respectiveinfusion time. An antibody solution used according to the invention canbe administered intravenously as a bolus injection of a concentratedsolution, or also in diluted form. The medicament can be diluted e.g.1:10 to 1:100-fold with physiological saline solution so as to providean infusion preparation.

By the term “antibody” antibodies of all types are to be understood, inparticular monospecific or polyspecific monoclonal antibodies, or alsochemically, biochemically or molecular-biologically prepared antibodies,or polyclonal antibodies having a certain specificity, e.g. an immuneserum or a fraction of an immune serum. The term antibody also includesneoepitope vaccines that are formed by the glycosylation of an antigenof a cellular surface protein with an antigen of an aberrantglycosylation.

An antibody utilized according to the invention preferably is a native,i.e. functionally active, antibody. This antibody preferably does nothave an attached label or other detection agent so as not to impair itsfunctionality. Native antibodies have the properties of the antibodiesnaturally occurring in patients. Native antibodies are heterotetramericglycoproteins composed of two identical light chains and two identicalheavy chains.

Yet also an antibody derivative may be used which preferably is selectedfrom the group of antibody fragments, conjugates, homologues orderivatives, or also complexes with additional effector functions. Inany event, it is preferred that the antibody derivative contains atleast parts of the Fab fragment, preferably together with at least partsof the F(ab′)₂ fragment, and/or parts of the hinge region and/or the Fcportion of a lambda or kappa antibody.

Furthermore, also a single-chain antibody derivative, such as aso-called single-chain antibody, can be employed according to theinvention. An antibody used according to the invention preferably is ofthe type of an immunoglobulin, such as an IgG, IgE, IgM, IgA or IgD.

According to the invention, the antibody binds directly to a tumor cellor to metastases, or micrometastases, respectively. A thereby formedimmune complex of the antibody is the prerequisite for the humoral andcellular activities of the immune system, expressed by anantibody-dependent cellular cytotoxicity (ADCC) and/or a complementdependent cytotoxicity (CDC) effector function. These effector functionsare determined by standard tests for the detection of function-blocking,receptor-blocking and prevention of adhesion.

High-affinity antibodies are preferred according to the invention. Inparticular, antibodies are used which bind with an affinitycorresponding to a dissociation constant of below a Kd value of 10⁻⁶mol/l, preferably less than 10⁻⁷ mol/l, most preferred 10⁻⁸ mol/l, orless.

Antibodies used according to the invention for the passive immunotherapymay be derived from a non-human species, such as a murine antibody. Yetit is expected that a recombinant, chimeric, as well as a humanizedantibody combined with murine and human components, or a human antibodywill be particularly tolerable for the administration on humans.

For the active immunization of cancer patients, according to theinvention the components a) and/or b) are formulated in immunogenicform, or as vaccines, respectively. Preferred are pharmaceuticalpreparations which contain a pharmaceutically acceptable carrier. Thelatter comprises, e.g., auxiliary substances, buffers, salts, preservingagents. The pharmaceutical preparations may, e.g., be used for theprophylaxis and therapy of cancer-associated conditions, such asmetastasis formation, in cancer patients. In doing so,antigen-presenting cells are specifically modulated in vivo or also exvivo so as to generate the immune response against the TAAs.

For the active immunization with the specific antigens or the antigencombination, respectively, of the kit according to the invention,usually a vaccine formulation is used which contains the immunogen—be ita natural TAA or its epitope, mimic or neoepitope mimic, or animmunogenic antibody—mostly only at low concentrations, e.g. in animmunogenic amount ranging from 0.01 μg to 10 mg. Depending on theimmunogenicity of the vaccination antigen which is, e.g., determined bysequences of a foreign species or by derivatization, or also dependingon the auxiliary substances or adjuvants, respectively, used, thesuitable immunogenic dose is chosen e.g. in the range of from 0.01 μg to750 μg, preferably 100 μg to 500 μg. A depot vaccine which is to bedelivered to the organism over an extended period of time may, however,also contain much higher amounts of vaccination antigen, e.g. at least 1mg to more than 10 mg.

The concentration will depend on the amount of liquid or suspendedvaccine adminstered. A vaccine usually is provided in ready-to-usesyringes or ampoules having a volume ranging from 0.01 to 1 ml,preferably 0.1 to 0.75 ml.

The vaccination antigen of a component of the inventive kit preferablyis presented in a pharmaceutically acceptable carrier which is suitablefor subcutaneous, intramuscular and also intradermal or transdermaladministration. A further mode of administration functions via themucosal pathway, e.g. vaccination by nasal or peroral administration. Ifsolid substances are employed as auxiliary agent for the vaccineformulation, e.g. an adsorbate, or a suspended mixture, respectively, ofthe vaccine antigen with the, auxiliary agent will be administered. Inspecial embodiments, the vaccine is presented as a solution or a liquidvaccine in an aqueous solvent.

Preferably, vaccination units of a tumor vaccine are already provided ina suitable ready-to-use syringe or ampoule. A stable formulation of thevaccine may advantageously be put on the market in a ready to use form.Although a content of preserving agents, such as thimerosal or otherpreserving agents with an improved tolerability, is not necessarilyrequired, yet it may be provided in the formulation for a longerstability at storage temperatures of from refrigerating temperatures upto room temperature. The vaccine according to the invention may,however, also be provided in frozen or lyophilized form and may bethawed or reconstituted, respectively, upon demand.

It has proved suitable to increase the immunogenicity of an antibodyused according to the invention by employing adjuvants. For thispurpose, solid substances or liquid vaccine adjuvants are used, e.g.aluminum hydroxide (Alu-Gel) or aluminum phosphate, growth factors,lymphokines, cytokines, such as IL-2, IL-12, GM-CSF, gamma interferon,or complement factors, such as C3d, further liposome preparations, oralso formulations with additional antigens against which the immunesystem has already generated a strong immune response, such as tetanustoxoid, bacterial toxins, such as Pseudomonas exotoxins, and derivativesof lipid A and lipopolysaccharide.

Epitopes of the suitable target antigens imitate or comprise primarilydomains of a natural, homologous or derivatized TAA. These arecomparable to the TAA at least by their primary structure and, possibly,their secondary structure. The epitopes may, however, also be completelydifferent in this respect from the TAAs and may imitate components of aTAA, primarily proteinaceous or carbohydrate antigens, respectively,simply by the similarity of spacial (tertiary) structures. Simply thetertiary structure of a molecule may, thus, form a mimic which triggersthe immune response against a certain TAA.

In a special embodiment, at least two equal or different epitopes of anadhesion protein, e.g. of a homophilic cellular membrane protein, suchas EpCAM, are provided, or imitated, respectively, on the inventivelyused antigen of component a). Thus, by the active immunization, aplurality of antibodies with a specificity for the same molecule, yetwith different EpCAM binding sites may be generated. Likewise, also anepitope of an antigen of component b) can be conjugated to one orseveral of the epitopes of the adhesion protein. An accordingly formed“combination antigen” thus comprises at least one epitope, fragment ormolecule of a cellular adhesion protein, such as EpCAM, and an epitope,fragment or molecule of an aberrant glycosylation, such as Lewis Y, onone single carrier. Such a combination antigen can imitate the cellulartumor antigens particularly well, and accordingly causes the desiredimmune response against the epithelial tumor cells.

For the vaccine formulation, also further known methods for conjugatingor denaturing vaccine components may be used so as to further enhancethe immunogenicity of the active substance. In addition, othersubstances, such as peptides, glycopeptides, carbohydrates, lipids ornucleic acids, yet also ionic groups, such as phosphate groups, orcarrier molecules, such as polyethylene glycol or KLH, can covalently bebound to inventively used vaccination antigens. These side groups maypossibly themselves represent epitopes of a tumor-associated antigen asdefined by the present invention. One example of a conjugatedvaccination antigen is the immunogenic antibody described in Austrianapplication A 744/2002 which is glycosylated with a Lewis Y antigenicstructure.

In an inventively used combination antigen, the various epitopestructures may be interconnected via a coupler. This coupler preferablyis a short bifunctional molecule, such as, e.g., N-hydroxysuccinimide.Likewise, the coupler may also be realized by a chemical compound largerthan a simple coupler molecule. It is always a prerequisite that thiscoupler will not have a negative influence on the immunogenic propertiesof the conjugate, i.e. that it by itself does not trigger anysubstantial immunogenicity. According to the invention, a coupler mayalso be produced by the chemical conversion of part of the vaccineantigen, or of the structure to be conjugated, respectively, practically“in situ”. This coupler produced at the epitope structure itself maythen be directly conjugated to the respective other binding partner(e.g. via the amine group of the lysine, via the OH groups, sulfurgroups, etc.).

According to a particular embodiment of the present invention, avaccination antigen is also provided in the form of its correspondingnucleic acid. The nucleic acid encodes for a corresponding epitope andmay optionally be directly inocculated as a “naked” nucleic acid so asto induce an immune response against the gene product in vivo.

Special embodiments of the vaccine according to the invention contain,in particular, anti-idiotypic antibodies as vaccination antigens, i.e.ab2 which are directed against the idiotype of a TAA-specific antibody(ab1). Anti-idiotypic antibodies provoke in vivo the formation of ab3antibodies which in turn also recognize the TAA of a tumor cell, bindthereto and lyse it accordingly. By way of example, an anti-idiotypicantibody against glycan-specific antibodies is used, such as ananti-idiotypic antibody which recognizes the idiotype of an anti-Lewis Yantibody, e.g. as described in EP 0 644 947.

These antibodies used according to the invention for the activevaccination are administered in small amounts only. Thus, e.g., nospecial side effects are expected, even if the antibody according to theinvention has been derived from a non-human species, such as, e.g., amurine antibody. It is, however, assumed that a recombinant, chimericantibody as well as a humanized antibody combined with murine and humancomponents, or a human antibody is particularly well tolerable for theadministration to humans. On the other hand, a murine portion in aninventive antibody on account of its foreignness may additionallyprovoke the immune response in humans.

The combination of the active and/or passive immunotherapy with adjuvanttreatment methods known per se is very usual. Among them are means ofradiotherapy or chemotherapy, such as the monotherapy or polytherapy.Because of the different mechanisms of action, the immunotherapypreferably is combined with the polychemotherapy. The combination of theactive immunotherapy of a cancer patient with a chemotherapy has beendescribed e.g. in Austrian application A 774/2002.

Agents preferably used for chemotherapy are alkylating pharmaceuticalpreparations. Thus, e.g., agents containing taxane, anthracyclines orplatinum are preferred. All the conventional preparations which areemployed for the various cancer treatments can be further combined. Thechemotherapeutic agents usually are administered intravenously orperorally.

The treatment according to the invention comprises both prophylactic andtherapeutic measures. The treatment is not only intended for the fieldof human medicine, but can also be used for the treatment and/or fordiagnosing forms of cancer of various mammals. Patients with primarytumors can be treated just as patients with secondary tumors. The cancertreatment is particularly targeted to the treatment of the “minimalresidual disease”.

A possible objective of treatment is the effective binding and reductionof tumor cells so as to prevent their dissemination as far as possible.Simultaneously, also particularly the disseminated tumor cells areattacked. The tumor cells or micrometastases detectable in blood, bonemarrow or organs are prophylactically prevented with the kit accordingto the invention, and significantly reduced therapeutically. Theformation of metastases is to be retarded in that their growth is atleast slowed down. Thus, by the immunotherapy according to theinvention, the relapse-free life span and, thus, also the total time ofsurvival of the patients can be extended. An indicator for the successof the treatment is the significant reduction of tumor cells in blood,serum or bone marrow.

The inventive kit of the antigens and/or antibodies also serves for theimmunologic determination of a cancer disease. Since the said targetantigens and the combination of the components a) and b) have provenparticularly telling for the further course of the disease and thesurvival time, the active substances can be used as diagnostic means. Inthis manner, an immunologically active “panel” is provided which servesto select suitable diagnostic antibodies or is directly suitable as animmune reagent for determining tumor cells of solid tumors, metastases,micrometastases or disseminated tumor cells.

For the diagnostic determination, samples of tumor tissue or bodyfluids, such as blood or bone marrow, are taken from tumor patients.These samples then are admixed with the immunoreagents according tocomponents a) and b) of the kit. A possible immune reaction is anindicator for the malignancy of the disease, and for the course of thedisease, respectively, and a certain prognosis.

Small tumors or a sample of tumor tissue are usually taken by biopsy.Samples of tumor tissue are also obtained by the partial or completeresection of solid tumors. On account of the tumor tissue taken, apathologist will then diagnose whether the tissue is from a benign tumoror from a malignant tumor. A diagnosis can be supplemented by theinventive method for immunohistochemistry, or it may be offered as astandardized alternative.

The method according to the invention of immunologically determining acancer disease with at least one selection of immunoreagents accordingto components a) and b) can be further employed for examining samples ofperipheral blood, bone marrow or fractions thereof. The advantageous useof a set according to the invention relates to the monitoring of cancerpatients during a therapy. In particular, immunoreagents are used fordetermining an antibody titer against the antigens of components a) andb). Appropriate immunoreagents are, however, also used for thequalitative or even better, the quantitative determination ofdisseminated tumor cells from body fluids of a cancer patient during atherapy. The reduction of the disseminated tumor cells is an indicationof the effectiveness of the therapy.

Monitoring during a cancer treatment preferably uses an inventivecombination of immunoreagents, not only for determining a correspondingantibody titer in the patients' blood, but also for determining theeffect on the tumor cells of solid tumors or on disseminated tumorcells.

Blood or serum samples from a cancer patient can be qualitatively and/orquantitatively examined for immune complexes with a diagnostic agentaccording to the invention. The analysis methods as such are commonanalysis methods with fractionation and enrichment of the immunecomplexes and/or immune reaction with a specific antibody, such as anantibody directed against the Fc portion of an antibody used fortreatment. If the used antibody has murine structures, the latter canalso be bound with an anti-murine antibody as in a capture step.

An immune reaction with the immunoreagents used according to theinvention can be detected by an appropriate labelling of one of thebinding partners from the immune complex. Usually an immunoreagent isprovided with a labelling. A further preferred variant of the diagnosticagent additionally comprises a reagent which reacts with the immunecomplex possibly formed. This reaction can then be made visible by meansof an appropriate labelling. The means for labelling immunoreagents orof reagents against the immunocomplexes are known to the person skilledin the art. Among them are fluorescent, chromogenic agents, radiolabelsor enzyme labels.

Reagents for determining the components a) and/or b) or for determiningtheir reaction products, respectively, or immune complexesadvantageously are immobilized on a carrier. Suitable carriers are,e.g., prepared microtiter plates, yet also carriers suitable forimmunoaffinity chromatography. The immune reagents advantageously aredirectly bound to column materials and bind the relevant antigens orantibodies from body fluids for determination thereof.

A preferred material for the inventively used diagnostic agent is, e.g.,immobilized EpCAM, a recombinant EpCAM being preferred. This agent willthen be combined with a further immobilized anti-idiotypic antibodyhaving a specificity for the idiotype of a Lewis Y antibody or with animmobilized Lewis Y antigen. The preferred combination is the serial orparallel purification of immunoreactants from a body fluid, e.g. serum,so as to quantitate them.

FIG. 1 shows the co-expression of EpCAM and Lewis Y antigens on tumortissue samples of female breast cancer patients, as well as theircorrelation with the survival time.

FIG. 2 shows Le-Y expression of different tumour cell lines, FACSresults. Le-Y was detected with IGN311 and visualized using aFITC-conjugated anti-human antibody.

FIG. 3 shows the Her2-neu expression pattern of the same cell lines alsoanalyzed by FACS using Herceptin® as detection antibody and the samesecondary antibody system. The major difference to the previous analyzedLe-Y expression pattern was that WM9, Kato III and SKBR5 were Her2-neunegatives. Only SKBR3 expressed this membrane-protein. Therefore, onlySKBR3 could be used despite its lower Le-Y expression density for ADCCexperiments.

FIG. 4 shows the results of the Antibody Dependent Cellular Cytoxicityassay: Lysis of SKBR3 cells was mediated by effecter cells of a healthydonor in combination with IGN311, Herceptin® and combinations ofconstant IGN311 concentrations (40, 100 and 800 ng/ml) with Herceptin®.

In the following, the immunohistochemical determination of relevantantigens on tissue samples of solid tumors will be described, as well asdiagnostic methods for the differentiated monitoring of immunospecifictumor markers. The following examples shall explain the presentinvention in more detail without, however, restricting it.

1. Immunohistochemical Examination of Microarrays

Tissue samples from solid tumors were prepared according to WO 0142796as microarrays which were examined immunohistochemically by means ofdifferent antibody preparations. The determined antigen structures ofthe microarrays were compared with the history of the donors of thetissue samples.

As antibody preparations, the following were used:

-   -   ESA (Novocastra) mouse monoclonal anti-EpCAM antibody;    -   BR55-2 (EP 0 285 059, ATCC HB 9324) mouse monoclonal anti-Lewis        Y antibody;

The immune reaction was determined by staining with a standardperoxidase-conjugated avidin-biotin system (Vector ABC-kit, Vector,PK-6100) with diamino-benzidase as chromogen (DAB, Bio Genex,HK-130-5K).

The degree of staining was evaluated according to a score (0, 1+, 2+,3+). In addition, the portion of positive cells was estimated. The tumorsamples were classified according to staining and to the portion of thestained cells as follows:

-   -   Negative: no staining    -   weak: 1+<70%, 2+≦20%    -   moderate: 1+≧70%, 2+≧20% and <80%, 3+<30%    -   strong: 2+≧80%, 3+≧30%

A. Determination of the EpCAM Expression on Tissue Samples of BreastCancer

The reaction of the microarrays with ESA was more pronounced in theadvanced state of the disease and with the degree of node formation. Amissing staining with ESA correlated with a favorable prognosis. It wasfound that ESA preferably binds to samples of neoplastic breast diseasesand tumors with an unfavorable prognosis. ESA, however, binds onlyweakly to normal breast tissue or to non-malignant breast diseases.TABLE 1 ESA immune reaction and tumor stage ESA Immune Staining Tumorstage N Neg % weak % moderate % strong % pT1 631 54.04  21.24% 14.58%10.14% pT2 856 43.57% 26.64% 14.37% 15.42% pT3 106 42.45% 24.53% 16.98%16.04% pT4 212 47.64% 22.64% 15.09% 14.62%p = 0.0078N . . . number of microarrays examined

TABLE 2 ESA immune reaction and tumor degree ESA Immune Staining Tumordegree N Neg % weak % moderate % strong % G1 429 64.57% 20.51%  8.62%6.29% G2 693 52.67% 25.11% 13.85% 8.37% G3 572 26.40% 26.75% 20.63%26.22% P < 0.0001

TABLE 3 ESA immune reaction and node status ESA Immune Staining Nodestatus N Neg % weak % moderate % strong % pN0 744 50.67% 20.97% 13.98%14.38% pN1 654 47.09% 25.38% 15.44% 12.08% pN2 105 33.33% 27.62% 18.10%20.95%P = 0.0115

B. Determination of the Lewis Y Expression on Tissue Samples of BreastCancer

Lewis Y is expressed on different samples of normal tissue and on tissuefrom neoplastic breast diseases. The reaction of the microarrays withBR55-2 was more pronounced with an advanced degree of tumor, yet did notcorrelate with the node status and tumor stage.

(Sauter et al., Abstract 107052, Meeting report, Eurocancer 2003) TABLE4 BR55-2 immune reaction and tumor stage Lewis Y Immune Staining Tumorstage N Neg % weak % moderate % strong % PT1 597 20.44% 30.15% 32.66%16.75% PT2 834 20.62% 31.65% 34.77% 12.95% PT3  95 12.63% 35.79% 41.05%10.53% PT4 206 20.39% 34.95% 31.07% 13.59%p = 0.2486

TABLE 5 BR55-2 immune reaction and tumor degree Lewis Y Immune StainingTumor degree N Neg % weak % moderate % strong % G1 412 24.76% 24.03%34.71% 16.50% G2 652 19.94% 31.90% 36.50% 11.66% G3 552 16.85% 38.22%29.53% 15.40%P < 0.0001

TABLE 6 BR55-2 immune reaction and node status Lewis Y Immune StainingNode status N Neg % weak % moderate % strong % PN0 713 21.04% 29.31%34.92% 14.73% PN1 629 20.67% 33.39% 31.64% 14.31% PN2  99 13.13% 39.39%34.34% 13.13%p = 0.2621

C. Determination of the EpCAM and Lewis Y Co-expression on TissueSamples of Breast Cancer

The co-expression of EpCAM and Lewis Y antigen on breast cancer tissueis found in many cases. The probability for the EpCAM specific immunestaining increases with the color intensity of the Lewis Y immunestaining. The co-expression is primarily frequently found in high-degreetumors, yet it is hardly associated with the tumor stage or node stage.

The prognosis for the survival time of the cancer patients is clearlydeteriorated if both antigens are expressed (p<0.0001), cf. FIG. 1 inthis context. TABLE 7 ESA and BR55-2 immune reaction and tumor stage ESAImmune Staining Tumor stage N Neg % Lewis Y % EpCam % both pos % PT1 56841.73% 32.92% 10.92% 14.44% PT2 799 39.55% 29.66% 15.64% 15.14% PT3  9338.71% 24.73% 16.13% 20.43% PT4 202 36.63% 33.17% 14.85% 15.35%p = 0.2482

TABLE 8 ESA and BR55-2 immune reaction and tumor degree ESA ImmuneStaining Tumor degree N Neg % Lewis Y % EpCam % both pos % G1 393 50.38%34.10%  8.91%  6.62% G2 627 44.66% 32.06% 11.32% 11.96% G3 536 25.19%27.24% 21.46% 26.12%p < 0.0001

TABLE 9 ESA and BR55-2 immune reaction and node status ESA ImmuneStaining Node status N Neg % Lewis Y % EpCam % both pos % PN0 679 42.56%27.98% 13.70% 15.76% PN1 605 38.84% 32.56% 13.06% 15.54% PN2  98 30.61%30.61% 16.33% 22.45%P = 0.1746

2. Monitoring the Immune Response to an Active Immunization

In a study regarding the active immunization of rhesus monkeys with avaccine based on a He2-Lewis Y neoglycoprotein (HE2LeY), preparedaccording to Austrian application A744/2002, the success of immunizationwas determined by way of the specific immune response.

A diagnostic agent was used so as to determine the titer of anti-EpCAMantibodies and anti-Lewis Y antibodies via the sequential immuneaffinity chromatography. The affinity chromatography was effected withthe assistance of an FPLC system. 1 ml of serum was diluted 1:10 inphosphate-buffered saline and applied to the first column. The boundfraction was recovered by elution with a glycine buffer or withammonium, and optionally applied to a second column. The immunoglobulincontent of the EpCAM and Lewis Y fractions was quantitated.

The following column materials were used for the diagnosticdetermination:

-   -   1. EpCAM-Sepharose: recombinant EpCAM bound to a CH-Sepharose 4B        column    -   2. SynsorbY: Lewis Y bound to a chromo sorb matrix (of Synsorb)    -   3. He2LeY Sepharose: He2LeY bound to a CH-Sepharose 4B column

Columns 1 and 2 were used both as sole diagnostic agents as well as inseries.

3. Determination of Disseminated Tumor Cells from Peripheral Blood

Disseminated tumor cells were determined from blood samples of tumorpatients in the following manner:

A. Tumor Cell Enrichment:

25 ml of peripheral blood were centrifuged at 1600×g for 20 min at 4° C.in an OncoQuick® tube (Greiner bio-one, Altmünster, Austria). The tumorcell-containing phase was transferred into a further centrifuge tube anda cell pellet was recovered by centrifuging. This pellet wasresuspended. The cellular portion of the suspension was centrifuged to aslide for microscopic examination. The slide was stored at −20° C. untilevaluation.

B. Tumor Cell Determination

A soltuion with fluorescence-labelled specific antibodies was applied tothe slide with the isolated and enriched tumor cells. After a period ofincubation of 30 min, the tumor cells labelled by the antibody bond werevisualized under the fluorescence microscope (Axioplan Zeiss, Jena,Germany) and counted. The content of tumor cells in the blood was thencalculated according to the factor of enrichment. The method wasvalidated by standard tumor cell suspensions.

IGN311, a humanized monoclonal anti-Lewis Y antibody, prepared accordingto EP 528 767, and HEA-FITC (Anti-EpCAM, Miltenyi, clone HEA125) bothconjugated with fluorescent proteins, are utilized as labelled, specificantibodies.

The effectiveness of the immunotherapeutic treatment of cancer patientsis proven by the detection of the tumor cells in peripheral blood. Thereaction of detected tumor cells with one of the two antibodies or withboth antibodies is differentiatedly evaluated within the scope ofmonitoring.

4. Selection of Antibodies with a Specificity for a Neo-epitope

BALB/c mice are immunized at first intraperitoneally and 4 weeks laterintravenously with cells of the SKBR3 breast cancer cell line. Spleencells are fused with suitable melanoma cells, and hybridomas areprepared. The monoclonal antibodies obtainable therefrom are examinedfor their specificity with a differential screening method. Theappropriate positive clones are used for production of the neo-epitopespecific monoclonal antibodies so as to produce antibody preparationsfor the immunotherapeutic treatment of tumor patients.

Differential screening is effected consecutively with the followingantigens:

-   -   A.Lewis Y-Her-2/neu receptor (SKBR3 cell membrane extract on        Western Blot)    -   B.Lewis Y (synthetic, Synthesom)    -   C.Her-2/neu receptor, recombinant

The neo-epitope antibodies bind only to antigen A, yet not to theantigens B or C.

EXAMPLE 2 Combinatorial Targeting of Tumor Cell Related Protein andOligo-saccharide Antigens

Two tumor associated antigens, the Le-Y glycosylation pattern and themembrane protein Her2-neu were chosen as simultaneous targets in ADCCexperiments. SKBR3 cell lines was selected between different other tumorcell lines over-expressing both antigens at highest densities. Serialdilutions of both antibodies were incubated with target cell line andPBMC's from a healthy donor. ADCC was analyzed using a ⁵¹Cr releaseassay for both antibodies, half time lysis were 1.1 μg/ml and 0.4 ng/mlfor IGN311 and Herceptin® respectively. 40, 100 and 800 ng/ml IG-N311were added as constant concentration to the serial Herceptin® dilution.Calculated Hill-slopes of lysis curves near their inflection pointincreased from nearly 1.0 for Herceptin® (same value for IGN311) tovalues of 2.6, 3.0 and 3.6 with raised con- centrations ofsimultaneously supplemented IGN311. Since these values exceed the effectof summarized individual effects, we could observe a synergistic effectby simultaneously targeting both tumor associated antigen structuresLe-Y and Her2-neu.

A purpose of a combined simultaneous targeting of different tumor cellrelated antigens is an enhancement of the involved individual mechanismsleading finally to more effective tumor cell lysis. Our chosentarget-structures in this example were the membrane anchored proteinHer2-neu and the membranous Le-Y glycosylation pattern. Both anti-genesare epithelial tumor-cell related and over-expressed on 70% of allepithelial tumors.

Material and Methods

IGN311 is a humanized anti-Le-Y antibody. Herceptin® is a commercialavailable antibody. Cell lines Kato III, WM9 and SKBR3 were purchasedfrom ATCC.

ADCC experiments were carried out as follows: 10⁷ SKBR 3 cells wereseeded in a 75 cm² flask and grown for 4 days. 110 ml of blood/platewere collected in Vacutainer CPT (Cell preparation tubes) from a healthydonor. PBMC's were isolated and washed twice with 0.1% glucose in PBScomplete, once spinning at 400 g, second time spinning at 75 g. 7.5×10⁵isolated cells were seeded per well. Cells were incubated overnight.Target cells were scraped the out of their flask and monodispersed inmedia. 25×10³ cells were seeded per well. They are resuspend them insmall amount of media (850 μl) and incubated with ⁵¹CrO₄ ²⁻ for onehour. Cells were washed twice afterwards with media and resuspended.Cell concentration was adjusted to [number of PBMC's per well] dividedby 30 in 100 μl to reach an effecter to target ratio of 30 to 1. Serialdilutions of the antibody(ies) to analyse were prepared in media. In anELISA-plate holding the PBMC's, 50 μl of antibody dilution (or media)were added, then 100 μl of cell suspension was supplemented. Suspensionis incubated for 18 hours in a CO₂ incubator. Supernatant is harvestedand the intensity of γ- radiation is counted. Two controls are preparedon the plate:

Maximum lyses, receiving (in this order) 50 μl of media, 100 μl of cellsuspension and 100 μl of SDS-solution; spontaneous release, receiving150 μl of media and 100 μl of cell suspension. Percent lyses in sampleis calculated with Formula 1:

Calculation of lyses in ADCC experiments:% lyses=((CPM_((sample))−CPM_((Spontaneous Release))/(CPM_((Maximum Lyses))−CPM_((Spontaneous Release))))×100

Results

One important requirement for the used cell line was the simultaneousover-expression of both antigens Le-Y and Her2-neu. Different cell lineswere screened for these parameters in FACS experiments. Kato III, SKBR3and SKBR5 were analyzed. FIG. 2 shows the FACS results concerning theLe-Y expression pattern. Humanized anti Le-Y antibody IGN311 was used todetect Le-Y glycosylation pattern. All three epithelial cancer lineswere compared to the melanoma cell line WM9. All three cell linesexpressed membranous Le-Y.

The results are shown in FIG. 2 shows the Le-Y expression of differenttumour cell lines, FACS results. Le-Y was detected with IGN311 andvisualized using a FITC-conjugated anti-human antibody.

FIG. 3 shows the Her2-neu expression pattern of the same cell lines alsoanalyzed by FACS using Herceptine as detection antibody and the samesecondary antibody system. The major difference to the previous analyzedLe-Y expression pattern was that WM9, Kato III and SKBR5 were Her2-neunegatives. Only SKBR3 expressed this membrane-protein. Therefore, onlySKBR3 could be used despite its lower Le-Y expression density for ADCCexperiments.

The results are shown in FIG. 3: HER2-neu expression pattern ofdifferent tumour cell lines, FACS results. Her2-neu was detected withHerceptin® and visualized using a FITC-conjugated anti-human antibody.

Antibody Dependent Cellular Cytotoxicity chromium release experiment,were carried out using SKBR3 target cells. They were incubated with ⁵¹Crfor one hour. Then, effecter cells from healthy donors (PBMCpreparation) were supplemented with serial dilutions of IGN311,Herceptin® and combinations of both antibodies. In the case ofHerceptin®, a serial dilution from 0.4 pg/ml to 40 ng/ml was analyzed,in the case of IGN311 from 2 ng/ml to 10 μg/ml. The combination ofHerceprtin® and IGN311 consisted of the identical serial Herceptin®dilution supplemented with three constant IGN311 concentration of 40,100 and 800 ng/ml.

SKBR3 cells were incubated over night (18 hours) with prepared effectorsuspensions (effecter cells with antibody solution).

Triplicate measurements were performed. Chromium release, a function ofcell lysis, was measured in supernatants using a gamma-radiationcounter. Lysis potential was calculated as described in Methods section.Data were fitted using a sigmoidal curve fit model (GraphPad Prismsoftware, Table 1) and results are displayed graphically in FIG. 4.TABLE 1 ADCC results: 4 Parameter sigmoidal curve fit results evaluatedby GraphPad Prism IGN311 IGN311 IGN311 IGN311 Herceptin ® 40 100 800BOTTOM 44.59 37.53 46.49 56.81 76.05 TOP 99.3 104.5 87.88 90.9 97.65LOGEC50 3.043 −0.3852 −0.4668 −0.515 −0.2398 HILL- 1.071 1.116 2.6422.998 3.592 SLOPE EC50 1103 0.4119 0.3413 0.3055 0.5758

FIG. 4 shows the results of the Antibody Dependent Cellular Cytoxicityassay: Lysis of SKBR3 cells was mediated by effecter cells of a healthydonor in combination with IGN311, Herceptin® and combinations ofconstant IGN311 concentrations (40, 100 and 800 ng/ml) with Herceptin®.

All curves showed in their maximal concentration range a lysis potentialof 89 to 104%. In the lower concentration range, bottom values of 38 and45% were obtained for Herceptin® and IG-N311. These values alsocorresponded to the ones measured for spontaneous lysis phenomenons inthis experimental setup (data not shown). IGN311 showed a half-timelysis by 1.1 μg/ml while the one of Herceptin® was in the range of 0.4ng/ml. For the combinatorial setup, constant IGN311 concentrations (40,100 and 800 ng/ml) were chosen in the dynamic range of the curve. TheHillslope value (Table 1) describes the slope of the fitted sigmoidalcurve near its inflection-point. This parameter is essential forcomparison of effects due to combination of both antibodies. IGN311 andHerceptin®, both supplemented alone to the effecter cells, showed ahillslope nearly equal to 1.0.

If both antibodies would react independently from each other, thecombination should not affect the resulting slope near the inflectionpoint of lysis curves although a shift to higher lysis potential shouldbe visible. If there would be an inhibition of both antibodies, theresulting slope near the inflection point of lysis curves would belowered.

As result of a synergical effect, hill-slope would increase. This is theobserve effect in our assay. The combination of Herceptin® with 40 ng/mlIGN311 resulted in a shift of the resulting slope near the inflectionpoint of lysis curves from 1.0 to 2.6. The supplementation with 100ng/ml and 800 ng/ml resulted even in slopes increased to values of 3.0and 3.6 respectively. A synergical effect may therefore have occurred.

Combination of IGN311 and Herceptin® leads to an enhanced lysispotential, grater than the effect that would have been observed by thesum of the individual components. This or an equivalent combinationtargeting both membrane anchored (glyco-)proteins and glycostructuresover-expressed in cancer cells may play an important role in passiveimmunotherapy of cancer.

EXAMPLE 3 Selection of a EpCAM-LewisY Neoepitope Specific Antibody

1. Immunization with tumor cells:

-   a) mice are immunized with EpCAM- and LewisY-positive Tumor tissue    according to standard methods. After preparation of hybridomas the    hybridoma clones are selected for neoepitope specificity by    differential screening with assays detecting the binding of the    hybridoma derived monoclonal antibodies to    -   A: EpCAM-and LewisY positive cells    -   B: normal epithelial cells (EpCAM positive, Lewis Y-negative        cells)    -   C: EpCAM-negative/Lewis Y-positive cells        Binding assays can be performed by eg. FACS analysis or        immuno-western blotting with electrophoretically separated cell        extracts.        Antibodies specific for the neoepitopes are binding in assay A,        but not in assays B and C.        2. Immunization with recombinant EpCAM and sugar: Alternatively,        purified or recombinant EpCAM can be used as well as synthetic        Lewis Y can be used for immunization and for screening.        Alternatively, screening for other aberrant glycosylation forms        such as sialyl-Tn,etc can be screened for in assay C. In this        case, Lewis Y glycosylation is replaced by the other sugar        moiety.        3. Alternatively, the neoepitope-specific ligands can be        selected by reacting libraries of ligands (such as scFv-, Fab-,        antibody-libraries, peptide libraries, random libraries using        other scaffolds than immunoglobulins).        4. Mimics for the neoepitope can be selected by using a        neoepitope-specific ligand as antigen. It can be used as an        immunogen or antigen to produce anti-idiotypic antibodies by        standard techniques such as hybridoma technique or screening        with specific binding-pair libraries (eg. antibody-, Fab-,        scFv-libraries) or by screening (panning) with other random        libraries (non-immunoglobulin scaffolds, for example “repeat        proteins” such as ankyrin, leucine-rich or armadillo repeat        protein, but also anticalins (Arne Skerra, in Recombinant        antibodies, 29-30 Apr. 2003, Meeting report; Andreas Plückthun,        in Recombinant antibodies, 29-30 Apr. 2003, Meeting report),        peptides).        The selected mimics are binding to the neoepitope-specific        ligand and can be displaced by natural, neoepitope containing        structures (eg cells expressing the neoepitope).

1. A kit for the combined use for the treatment of cancer patients,which set comprises the following components: a) an antigen comprisingat least one epitope of a cellular surface protein, or an antibodydirected against the cellular surface protein, and b) an antigencomprising at least one epitope of an aberrant protein glycosylation, oran antibody directed against the aberrant protein glycosylation.
 2. Akit according to claim 1, characterized in that the components a) and b)are contained in one pharmaceutical preparation each or in a singlepharmaceutical preparation suitable for immunotherapy.
 3. A kitaccording to claim 2, characterized in that the pharmaceuticalpreparation is formulated as a vaccine.
 4. A kit according to claim 2,characterized in that the pharmaceutical preparation is formulated as anintravenously tolerable product.
 5. A kit according to any one of claims1 to 4, characterized in that the antigen of component a) represents anepitope of a cellular adhesion protein, in particular of a proteinselected from the group of EpCAM, NCAM and CEA.
 6. A kit according toany one of claims 1 to 4, characterized in that the antigen of componenta) is an epitope of a surface receptor, in particular a receptormolecule selected from the group of the EGF receptor family, CD55receptor, transferrin receptor and P-glycoprotein.
 7. A kit according toany one of claims 1 to 6, characterized in that the antigen of componentb) represents an epitope of a carbohydrate selected from the group ofLewis antigens, in particular Lewis y and/or Lewis b, sialyl-Tn andGlobo H.
 8. A kit according to any one of claims 1 to 7, characterizedin that the antigen of component a) represents an epitope of the EpCAMmolecule or of the Her-2/neu receptor, and the antigen of component b)represents an epitope of the Lewis Y molecule.
 9. The use of a kitaccording to claim 1 for preparing a diagnostic agent for theimmunologic determination of tumor cells of a solid tumor ordisseminated tumor cells of a cancer disease.
 10. The use according toclaim 9, characterized in that the determination is carried out withinthe scope of the treatment of cancer patients.
 11. The use according toclaim 9, characterized in that tumor cells from samples of peripheralblood or bone marrow are determined.
 12. The use according to claim 9 or10, characterized in that an antibody titer against the antigens of thecomponents is determined.
 13. The use according to claim 12,characterized in that the determination is carried out for monitoring atreatment of a cancer patient.
 14. A method for immunologic selection ofa tumor-specific target antigen or of antibodies directed against thetarget antigen by using a kit according to claim 1, characterized inthat the antigen is a neoepitope which is formed by the glycosylation ofan antigen of component a) with an antigen of component b).
 15. Apreparation of an antigen which comprises a neoepitope or its mimic,obtainable by a method according to claim
 14. 16. A preparationaccording to claim 15 wherein the antigen is a naturally occurringantigen or a fragment thereof.
 17. A method according to claim 14,characterized in that an antibody directed against the neoepitope isselected and prepared by using a kit according to claim
 1. 18.Preparation of an antibody with specificity for a neo-epitope,obtainable by a method according to claim
 17. 19. A diagnostic agentbased on a kit according to claim 1, characterized in that it contains areagent for determining an immune reaction with components a) and b), orwith antibodies against these.
 20. An agent according to claim 19,characterized in that the reagent is labelled with a fluorescent agent,a chromogen, a radiolabel or an enzyme.
 21. An agent according to claim20, characterized in that the reagent is immobilized on a carrier. 22.An agent according to claim 21, characterized in that the carrier is amatrix for immunoaffinity chromatography.